JP2022520996A - Modular scanning confocal optical profile microscope with digital image formation processing - Google Patents

Abstract

SOLUTION: In one embodiment, a beam steering means arranged so as to direct an electromagnetic radiation source on a non-identical line with respect to an optical axis of a focusing lens means configured in a modular confocal optical microscope. An electromagnetic radiation source oriented on a focused non-identical line is used as the basis for image formation of one or more target sites in the specimen with beam steering means and one or more in the specimen. At least one aligned detector configured on a composite confocal plane along a beam path to multiple target sites, using an image formation basis to determine the focus and image quality of one or more target sites. To maintain, alignment is further configured to provide auto-correction information, as well as provide high throughput to form a synthetic aperture in the pixel range from N = 2x2 array to N = 21x21 array. A modular confocal microscope comprising the detector is disclosed. [Selection diagram] FIG. 1A

Description

● Cross-reference of related applications This application is a provisional U.S. patent filed on February 26, 2018 under the name of "Using persistent photoconductivity to write a low-resistence pass in SrTIO3" under Article 119.35 of the U.S. Patent Act. Claiming the priority benefit of Application No. 62 / 635,134, the provisional application is incorporated herein by reference in its entirety.

● Government interests The present invention was made with the assistance of the national treasury under the DMR1561419 awarded by the National Science Foundation. Government has certain rights in the present invention.

Embodiments herein relate to rapid and highly accurate image formation and / or detection of artifacts using a confocal microscope. In particular, the arrangement of confocal microscopes disclosed herein, and the present embodiments using the technique, are directed to wide-field image formation, artifact detection, and adjustment of materials exhibiting permanent photoconductivity.

● Description of related fields Confocal laser scanning microscopy (CLSM) is a mature technique widely used in life sciences. Confocal laser scanning microscopy has been used to obtain detailed information about cells, tissues, microbial biofilms, and brain sections. Labeling cells with fluorescent dyes is a preferred technique for providing contrast in optical images. In addition to biomedical studies, confocal microscopy has clinical potential to detect corneal diseases, skin cancer, and other conditions.

A useful aspect of CLSM is the use of such instruments to dispel out-of-focus light and, as a result, to obtain crisp, high-resolution images. A large area of the specimen is illuminated by a wide-field fluorescence microscope. Photons emitted from one area of the specimen are scattered or diffused to another location and are collected by the camera. The result is a "haze" that obscures important details. CLSM solves the problem of fluorescence blur by, for example, reconstructing an image from the collected data scanned by a laser that traverses the specimen from point to point. Moreover, for three-dimensional (3D) applications, out-of-focus features add blurry blur to each image plane, resulting in reduced contrast.

High quality images produced by such techniques typically cost more than $ 200,000 in a single microscope. The main cost in scanning arrangements is a scan / descan system that guides the laser to the sample and directs the emitted light back to the detector. Optical components must be placed within tight tolerances. In addition, the detector (typically a photomultiplier tube, or PMT) requires a high voltage power supply. High-performance confocal microscopes are familiar in biomedical research facilities, but are out of reach for many individual researchers, small businesses, or groups of university students. Moreover, the microscope is not portable, limiting its use in the field or in confined spaces.

In addition, CLSM is inherently slow because a small focused laser beam builds a digital image every time it scans the specimen. For example, in such an optical scanning device as is currently practiced, the acquisition time is approximately 15 minutes for a 200 × 200 image. In general, this is not a problem for the production of spectroscopic maps that require a long integration time to obtain a sufficient signal-to-noise ratio (S / N ratio) in most biological imaging applications. However, such scan times are not desirable in situations where high throughput is required.

For background information on the confocal laser scanning microscope system, see Mathew D.C. It is described in Patent Document 1 filed on September 5, 2013 under the name of "Digital Confocal Optical Profile Microscope" by McCluskey and is claimed. It states, "... certain confocal microscopes can have higher optical resolution than wide-field microscopes. However, such confocal microscopes have some drawbacks, eg, such confocal microscopes. A pinhole in a confocal microscope acts as an analog computer that applies a circular aperture function to the optical signal from the sample, which allows the focused optical signal to pass through but may carry a lot of information about the sample. Reject unfocused signals that may not be possible. As described in more detail below, some embodiments of the technique monitor substantially the entire beam profile of the optical signal from the sample. The beam profile created can be modeled to provide additional and / or more accurate information about the sample than existing confocal microscopes. "

Another technique for addressing the time acquisition and throughput issues mentioned above is the Scanning Disk Confocal Microscope (SDCM), which focuses light on an aligned rotating pinhole disk array. Use a rotating microlens array. Since the coupled microlens array and pinhole array disk are rotated at about 5,000 to 10,000 rpm, the field of view (FOV) is illuminated by the focused laser beam spot for some time. In some applications, fluorescence is excited and at the same time an image is formed from multiple points on the sample.

However, the problem is that the rotation of the rotating disk and the exposure time of the camera do not work together to receive the same number of laser beam exposures during the camera exposure time at all positions in the FOV, resulting in an image of the artifact. Exists. Moreover, the presence of multiple pinholes on the pinhole array disk causes unfocused light from other focal planes to enter adjacent pinholes. This impairs the z-axis resolution and makes the SDCM inferior to the CLSM in terms of z-axis resolution. Another undesired aspect of CLSM is that multicolor applications can be complicated by the need for multiple cameras, dichroic mirrors, and filter wheels. A single camera can be employed and the filter wheel can in turn excite different phosphors and quickly switch to detect, which hinders fast acquisition of multiple probes.

For background information on the scanning disk confocal microscopy system, which discloses a microlens array disk paired with a pinhole array disk to focus light to a pinhole, see Fabro et al. Explained in Patent Document 2 filed under the name of "Confocal Microscope" on July 23, 1991, and claimed. To that end, the present invention provides a confocal microscope including a light source that irradiates a part of a test piece with light and an aperture plate arranged between the light source and the test piece. The aperture plate is a light source. Includes an opening to allow a portion of the light emitted from the specimen to pass over a portion of the specimen. A means for focusing a portion of the light over the opening of the aperture plate is a light source. Placed between the aperture plate. An objective means for focusing the light that has passed through the aperture onto the specimen is between the aperture plate and the specimen in order to illuminate a part of the specimen. Arranged. A means of optically connected to a portion of a light-irradiated specimen is provided for collecting an image produced by light shining on a portion of the specimen. By focusing the portion over the opening of the opening plate, most of the light directed towards the opening plate passes through the opening plate and, as a result, increases the efficiency of light transmission through the opening plate. In a preferred embodiment of the invention, the confocal means includes a Fresnel zone plate. In an alternative embodiment, the confocal means includes a Fresnel lens, a microscope, or a microfunnel light source. " It is described as.

Additional background information regarding confocal microscopy systems disclosing microlens arrays connected to pinhole array disks can be found in Hell et al. Explained and claimed in Patent Document 3 filed on November 3, 2004 under the name "Two Microlens Arrays And a Pinhole Diaphragm Array". It includes a microlens array with multiple microlenses that divides the light beam of the illumination light into multiple converging partial light beams that simultaneously illuminate the sample at several measurement points. A beam splitter that separates the beam path of the sample light, which is derived from the beam path of the illumination light and the light irradiation of the sample and is captured in the opposite direction with respect to the illumination light, and multiple pinholes arranged in the beam path of the sample light. An additional microlens array with a pinhole diaphragm array that has a diaphragm and corresponds to the microlens of the microlens array that divides the illumination light, and a plurality of microlenses that correspond to the microlenses of the microlens array that divides the illumination light. The microlens of the microlens array that divides the illumination light and the microlenses of the further microlenses are arranged in the beam path of the sample light. The beam splitter is the microlens array that divides the illumination light. And the additional microlens array, and the pinhole diaphragm of the pinhole diaphragm array is not arranged between the microlens array that divides the illumination light and the additional microlens array. " Are listed.

Another technique for addressing the time acquisition and throughput problems described above is a technique called a lattice confocal microscope, often called a Structured Illumination Microscope (SIM), which is a wide-field method without lenses or scanning. ,. Such systems / methodologies rely on the placement of the mobile grid pattern in the excitation light path between the light source and the sample in order to project the grid pattern onto the image plane. Three images of the fluorescent sample are recorded using a grid of horizontal lines at three different positions. The calculation removes the out-of-focus light, leaving behind a single image containing only the in-focus information. A "regular" wide-field image can also be calculated by combining the three images together.

However, the limitation of such a method is slow, and if the sample is thicker, the grid pattern required for the calculation can be lost in the blur, often due to the latent residual grid pattern in the image. Such methods / systems tend to be noisy as the artifacts remain and the images are subtracted. Moreover, the method relies on three consecutive images to generate a single confocal slice, so the methodology is that the sample moves in time between each of the three image frames. Not suitable for rapid, dynamic, live cell imaging, as possible.

U.S. Pat. No. 9,891,422 U.S. Pat. No. 5,162,941 U.S. Patent Application Publication No. 2005/0094261 (A1)

Therefore, there is a need for a portable confocal light microscope system that has new aspects other than those described above and is capable of providing high quality images with a wide field of view at high throughput. In addition, the embodiments and corresponding methods herein are much cheaper than current systems and offer the benefits of confocal microscopy. It is portable, occupies a small area, and minimizes the number of moving parts, unlike the high-end CLSM. The embodiments of the specification are directed to such needs.

In the first aspect, in a modular confocal microscope, the electromagnetic radiation source is directed on a non-identical line with respect to the optical axis of the electromagnetic radiation source and the focusing lens means configured in the modular confocal microscope. A beam steering instrument located in a beam steering instrument that is focused and oriented on a non-confocal line is used as the basis for image formation of one or more target sites in a specimen. Means and at least one aligned detector configured on a composite confocal plane along the beam path to one or more target sites in the specimen, one or more using an image forming basis. Further configured to provide auto-correction information to maintain focus and image quality at the target site, constructing a synthetic aperture in the pixel range from the N = 2 × 2 array to the N = 21 × 21 array. An aligned detector that provides high throughput and at least one aligned detection configured to identify the two-dimensional and / or three-dimensional characteristics of one or more target sites of the specimen. A modular confocal microscope comprising a processor connected to the vessel is disclosed.

In the second aspect of the present invention, the electromagnetic illumination source and the microlens array arranged to receive the electromagnetic illumination source, and the received electromagnetic illumination source is adjacent to each other along the first beam path by the microlens array. A microlens array that is reconstructed into multiple subbeams that run on it, and an optical lens means that is configured to collect and focus multiple subbeams on multiple target sites, with multiple target sites. An optical lens means that collects and focuses the subbeams and then collects the induced reflected light and / or the emitted light from each of the plurality of target sites and directs them along the second beam path. The resulting reflected light and / or the emitted light provided by the signals from the focused sub-beams, which are configured along the second beam path, image the resulting multiple sites. At least one aligned detector configured such as that provides high throughput constituting a synthetic aperture in the pixel range from N = 2 × 2 arrangement to N = 21 × 21 arrangement. Modular micro with a detector and a processor connected to at least one aligned detector configured to identify the two-dimensional and / or three-dimensional characteristics of the target site. A lens array cofocal microscope is disclosed.

In a third aspect of the present invention, a modular confocal microscope is incorporated to simultaneously image and adjust one or more target sites in a test piece exhibiting permanent photoconduction. It is a beam steering means arranged so as to orient the electromagnetic radiation source on a non-alignment line with respect to the optical axis of the electromagnetic radiation source and the focusing lens means configured in the modular confocal microscope, and is focused. Confocal electromagnetic radiation sources directed to the beam steering means and up to one or more target sites in the specimen, which are used as the basis for imaging one or more target sites in the specimen. At least one aligned detector configured on a composite confocal plane along the beam path to automatically maintain focus and image quality at one or more target sites using an image formation basis. Aligned detectors and tests that are further configured to provide correction information and provide high throughput that constitutes a confocal aperture in the pixel range from N = 2 × 2 arrays to N = 21 × 21 arrays. A step of incorporating, comprising a processor connected to at least one aligned detector configured to identify two-dimensional and / or three-dimensional characteristics of one or more target sites of the body. A step of selecting a predetermined emission spectrum provided by a light source, a step of irradiating the initial contact point through the confocal geometric structure of a modular confocal microscope, and a test for irradiation while maintaining the irradiation. Disclosed is a method of preparing a material using a modular confocal microscope, including the step of paralleling the specimen to plot the path of body exposure.

Accordingly, as disclosed herein, embodiments provide high quality images with a wide field of view at high throughput. In addition, the embodiments and corresponding methods herein are much cheaper than current systems and offer the benefits of confocal microscopy. It is portable, occupies a small area, and minimizes the number of moving parts, unlike the high-end CLSM.

An embodiment of a modular microlens array confocal optical microscope (COP) system with digital image processing is shown. A fluorescent laser-guided spot imaged by a CCD array acting as a synthetic aperture is shown. The concept of a microlens array that divides a light source (ie, a laser) into an array of focused spots on a specimen is shown. The fluorescent spots generated from the microlens array image formed by the CCD array are shown. The fluorescent spots generated from the microlens array converted to the intensity value are shown. A scan of a laser array illumination spot is shown. The state of the image after covering the entire test piece is shown. FIG. 3A shows an additional embodiment of a modular scanning confocal optical microscope (COP) with digital image processing capability. FIG. 3B shows an off-axis in-focus / image-forming technique to allow any of the microscopy embodiments herein to stay in focus while simultaneously image-forming / processing. FIG. 3C shows the general principle of translation and defocus (signal intensity reduction) of a CCD array image based on the off-axis focusing / image forming technique shown in FIG. 3B. The measured axial response curve in the optical fiber detection curve and the CCD-based detection pixel cropping size are shown. An image of a chip carrier test piece obtained by optical fiber confocal scanning is shown. In particular, the image is an image of a 100 μm × 100 μm area on a 3 mm × 3 mm chip carrier obtained by plotting the intensity map obtained from fiber optic detection. Fiber image formation for comparison with CCD-based detection results is shown. The CCD-based detection result of 2 × 2N pixel crop size is shown. The CCD-based detection result of 6 × 6N pixel crop size is shown. The CCD-based detection result of 11 × 11N pixel crop size is shown. The detection result of the 21 × 21N pixel crop size CCD base is shown. The CCD-based detection result of 31 × 31N pixel crop size is shown. The CCD-based detection result of 41 × 41N pixel crop size is shown. The CCD-based detection result of 51 × 51N pixel crop size is shown. The CCD-based detection result of 101 × 101N pixel crop size is shown. Shown is an intensity profile of a horizontal line penetrating a polar marker for indicating the horizontal width of the polar marker O at a particular Y position and an electrode (AB) of various pinhole sizes. Shown is an intensity profile of a vertical line penetrating an isopolar marker to indicate the vertical width of the polarity marker O at a particular X position and electrodes (AB) of various pinhole sizes. The full width profile of the electrodes over the entire X and Y ranges measured in the case of optical fiber and the case of a CCD with a pinhole size of 21 × 21 pixels is shown. The resistance of the STO sample after exposure to 405 nm light is shown. The measurement was performed in the dark. The resistance of the sample before light irradiation was about 1 MX. The absorption spectrum of the SrO powder mixed with KBr after 6 days of air exposure is shown. Freshly opened SrO powder mixed with KBr was used as a reference. The low temperature (125K) IR spectra of STO annealed with 0.1 g of Sr (OH) ₂ under vacuum, 1/2 atm hydrogen gas, or oxygen gas are shown. The spectrum before light exposure was used as a reference for the absorbance plot. The low temperature (125K) IR spectrum of the STO annealed in steam is shown. The spectrum before light exposure was used as a reference.

Unless implicitly, clearly understood, or stated in the description of the invention herein, words appearing in the singular include the plural, and words appearing in the plural are the singular. It will be understood to embrace the shape. Moreover, unless implicitly, clearly understood, or stated, it is possible to list for any given ingredient or embodiment described herein. It will be appreciated that various candidates or alternatives may generally be used individually or in combination with each other. Moreover, it should be understood that the figures shown herein are not necessarily drawn to a constant scale and that some of the elements may be drawn solely for the sake of clarity of the invention. Is. In addition, reference numbers may be repeated between various figures to indicate corresponding or similar elements. Further, unless implicitly, clearly understood, or stated, it will be understood that any list of such candidates or alternatives is merely exemplary and not limiting. .. In addition, unless otherwise stated, numbers representing quantities such as raw materials, ingredients, reaction conditions, etc., as used herein and in the claims, should be understood as being modified by the term "about". ..

Thus, unless indicated to be in conflict, the numerical parameters described herein and in the appended claims may vary depending on the desired properties to be obtained by the subject matter presented herein. Is. At the very least, it does not seek to limit the application of the doctrine of equivalents to the claims, at least for each numerical parameter, taking into account the number of significant digits reported, and by applying the usual rounding techniques. Should be interpreted. Although the numerical ranges and parameters that describe the broad range in the subject matter presented herein are approximations, the numerical values described in the particular embodiment are reported as accurately as possible. However, any number essentially contains certain errors that inevitably occur as a result of the standard deviations found in each test measurement.

● Overview A useful embodiment disclosed herein is a modular scanning confocal microscope that uses a CCD camera to replace physical pinholes with materials science applications. The data collected by the CCD is processed to obtain an image of the test piece. A virtual pinhole is created by selecting the number of effective pixels in the recorded CCD image.

Apart from the configuration of conventional confocal microscopes, another useful embodiment disclosed herein uses a CCD camera to replace physical pinholes with material science applications. However, it also incorporates a fixed microlens array to divide the laser into an array of focused spots on the sample, for example to enable wide-field scanning / image formation at high throughput. It is also a modular confocal optical microscope.

Another novel aspect of the embodiments herein is that, for any of the disclosed embodiments, the input light source (eg, laser light source) used for light irradiation / image formation is investigated / To initiate image formation of the specimen / sample to be adjusted, it can be oriented slightly non-aligned with respect to the optical axis of the objective lens (eg, up to about 1 degree). With such a configuration, the image is originally formed as such, but then the translated and / or tilted sample plane is as shown in the number of pixels in the configured CCD array (x, The result is an image showing defocus and translation at y). Such novel techniques are beneficially used by the microscope configurations herein to maintain focus and mitigate data acquisition imperfections. In particular, it is scanned if, for some reason, the first image-formed site in the sample moves in parallel (eg, a plane that moves in the Z direction with respect to the in-focus object of the confocal microscope) or during scanning. If the surface of the area is not uniform, such events are automatically corrected to maintain proper focus and, as a result, often due to defocus while the microscope is operating. Images can be formed in real time by monitoring the offset and the decrease in signal strength.

Accordingly, in all disclosed embodiments, the methodologies and corresponding systems disclosed herein represent a bridge between a wide-field microscope and a confocal microscope. And a useful embodiment is not only the reflection image formation, but also a specimen such that hard pinholes are usually collected by an aligned CCD array operated at a location located in a typical confocal geometry. Also includes image formation of induced fluorescence emitted by.

In addition, the configurations herein are beneficial for 2D image formation, while embodiments herein are also capable of 3D optical sectioning, in which case with a thin, uniform layer. The rendering of the image can be digitally stacked on the 3D display of the desired object. Examples of such methodologies using embodiments herein are images of biological specimens that have been treated with fluorescent dyes or that have been genetically modified to fluoresce under appropriate lighting. The ability to form is mentioned.

Image formation by reflection and fluorescence is a preferred embodiment herein, as well as for optical image formation and material processing / adjustment herein without departing from the spirit and scope of the present invention as a whole. It should also be understood that embodiments of this specification are available. For example, embodiments herein show permanent photoconduction (discussed in detail below) while simultaneously imaging the target to ensure that the appropriate photons are recorded in the area. Certain wide bandgap (> 2.9 eV) materials can be processed / adjusted. Such permanent photoconducting materials that last for at least one year and often last forever include strontium titanate (SrTiO ₃ or STO), but various other oxide materials such as, for example. , But not limited to these, zinc oxide (ZnO) and the like can also be included. III-V semiconductor materials such as gallium nitride (GaN), gallium nitride indium arsenide (GaInNAs), and gallium arsenide (GaAs) can also be adjusted / processed as disclosed herein.

● Detailed Description Here, referring to the figure again, FIG. 1A is a modular microlens array cofocal optical microscope (COP) with digital image processing, generally represented by the number 100, according to an embodiment of the present invention. It is a general schematic diagram of a system. As shown in FIG. 1A, the COP microscope system (100) of FIG. 1A has an electromagnetic source 2, at least one lens system 4, a first steering mirror 6, a dichroic mirror 8, a relay lens 12 (tube lens), and an objective lens. 14, an optical filter 17, and a detector 18, a two-dimensional charge coupling detector (CCD) for including a complementary metal oxide semiconductor (“CMOS”) detector (eg, 8 bit dynamic range, pixel size). Includes a 5.6 μm × 5.6 μm DMK 640 × 480 pixel array), as well as a controller / processor 102 to assist the user in operating the system 100. As an embodiment of a practical example, the microscope is about 1.5 feet (46 cm) high and occupies less than 1 square foot (30 x 30 cm ² ). As another arrangement, red LEDs are used for wide range transmission (not shown). The beam splitter 318 is an uncoated optical component and a coated splitter (eg, an electron beam-deposited layered coating splitter or any layered coating optic to provide multi-wavelength selectivity (dycroic)), a cube. It can include splitters, half mirrors, prisms, and / or other suitable components.

In an exemplary embodiment, the controller / processor 102 communicates with a Microlens Array Confocal Optical Microscope (COP) 101 (as also indicated by a double arrow). The controller / processor 102 is a network server, desktop computer, and / or various circuit components of known types for providing device control, data analysis, etc. for the configuration of the embodiments disclosed herein. Other suitable computing devices, such as, but not limited to, general-purpose or dedicated processors (digital signal processors (DSPs)), firmware, software, and / or hardware circuit components. Can include.

In the use of such exemplary computing devices, as disclosed herein, the individual software modules, components, and routines incorporated include C, C #, C ++, Java, and / as source code. It should also be noted that it can also be a computer program, procedure, or process written in another suitable programming language. Image processing and data analysis are often performed in MATLAB® and Origin®. A computer program, procedure, or process can be compiled into intermediates, object code, or machine code and presented for execution by any of the preferred example computing devices described above. Various implementations of sources, intermediates, and / or object codes, as well as related data, include one or more computer-readable storage media, such as read-only memory, random access memory, magnetic disk storage media, optical storage media. It may be stored in a flash memory device and / or other suitable medium. According to aspects of the invention, computer readable media means known media as understood by those skilled in the art, which can be read (scanned / sensed) by a machine / computer / processor and the machine / computer / processor. Has hardware and / or coded information provided in a form that can be interpreted by the software. As used herein, it should also be understood that the term "computer-readable storage medium" excludes the propagating signal itself.

All of the components listed above are often intended to protect certain components from contamination and / or to be coupled to have an integrated compact system that is easy to transport and install. Therefore, it should be noted that it is housed in or connected to the housing 15. The component-by-component description of System 100 of FIG. 1A is useful for illustrative purposes, as well as in the field of light microscopy / spectroscopy when using the particular embodiments disclosed herein. Other alternative commercial and alternative commercial components with various other components illustrated (see description in FIG. 3A for exemplary components) known and understood by those of skill in the art. It should also be noted that it should be understood that it is also possible to incorporate custom configurations.

More specifically with respect to those components mentioned above, the electromagnetic source 2 is often a substantially monochromatic source, more often a laser source (4 below herein), eg, Consists of a wavelength variable laser light source, etc., but has a light emitting diode, halogen lamp, mercury lamp, and / or the desired frequency band for a particular application (image formation, fluorescent emission, etc.) and the required intensity. It can also be any other suitable type of light source (eg, a white light source) configured to produce an illumination beam configured to provide (eg, by filtering). In exemplary non-limiting fluorescence applications, the laser light source 2 is often configured to be received by a lens system 4 which is often a microlens array (4 below herein). A laser beam (eg, a 4.5 mW laser diode at a wavelength of 532 nm), but not always.

By analyzing the CCD image, as shown in FIG. 2C, the emission characteristics generated from each laser spot are often converted into an intensity value of 52 (pixels). Further, FIG. 2D is an appropriate step of scanning the steering mirror 6 (eg, a piazo scanning mirror), or the xyz stage 19 shown in FIG. 1A, which is a sub-increment of the pitch of the sub-lens 4'. Shown is a laser array 54 scanned by either mode-operated. The laser array scans the specimen so that each (x, y) point is covered. The approach to this exemplary non-limiting example allows for acquisition times comparable to high-end confocal systems while efficiently performing approximately 100 pinhole / detector (for 4x5 grids) measurements. do.

However, it should be noted that additional arrangements can include a piezoelectric objective scanner for vertical scanning and / or a piezoelectric nanopositioning stage for horizontal scanning and / or z-axis scanning, if desired. sea bream. Further, the xyz stage 19 is often a motorized linear stage configuration (eg, a stepper motor driven linear stage), but due to translational movement of a large area, it is motorized positioning. Manual positioning is also available in combination with. An example stepping operation using a stage known and understood by one of ordinary skill in the art is approximately 0.1 micrometer laterally, but beam conditioning, collection, and focusing optics such as relay lens 12, objective lens 14. , And any larger step increment is also possible, depending on the illumination characteristics provided to the specimen 1 by the microlens 4'lens array (eg, array 4'diameter, f #, etc.). FIG. 2E shows the results of the surface of the entire test body 1 which is covered as a result.

The array of unique targets illuminated with light then provides optical information (eg, image formation information by reflection (ie, forming an image of the electromagnetic source 2 laser spot), and / or fluorescence), which returns. Along the path 23, the objective lens 14, the relay lens 12, and the dichroic splitter 8 configured to transmit the wavelength of interest pass through, often one or more windows in the housing 15 (not detailed). ), And then directed to each pixel on the CCD detector 18 through one or more bandpass filters 17.

The charge-coupling detector (CCD) 18 comprises a detection area designed to be equal to or greater than the cross-section of the received beam signal, or an area comprising a plurality of beam signals in the photodetector 18. Note that it can be done. In many cases, the area itself for the detector is at least 5 times larger than the area of the received beam signal or multiple beam signals. The detector 18 is configured with a motorized control (eg, stepper control (not shown)) to move along at least one of the x-axis, y-axis, or z-axis, thereby. The detector 18 can maximize the image formation of the test piece 1 in the confocal method. However, it can also be placed away from the focal imaging plane designed by System 100 for other desired techniques. As a result, the percentage of detection areas of the photodetectors 90 and 70 filled with the signal beam is set to target values (eg, about 0.01, about 0.02, about 0.03, about 0.04, about 0. It may be increased to 05, about 0.06, about 0.07, about 0.08, about 0.09, or about 1.0).

During operation, the controller 102 causes the illumination source 2 to generate a beam 3 for illumination (either by the user or under automatic control). The microlens array 4 is then configured to receive the beam 3 of the laser light source 2, as generally shown in FIG. 1A. The microlens array 4 is often composed of a two-dimensional (2D) lattice of the sublens 4'(microlens), more often a 4x5 lattice, in which case the sublens 4'(. Each of the lenslets) changes the initial wavefront of the laser light source 2 into an array of spots (eg, 52). The actual dimensions of the individual sublenses 4'include at least one dimension (eg, diameter) ranging from a few micrometers to a few tens of micrometers.

To better understand the role of the microlens 4 in the operation of the system 100, FIGS. 2A, 2B, 2C, 2D and 2E show this aspect. In particular, the grid pattern of the microlens array 4 divides the laser into an array of corresponding spots (subbeam 50 (see FIG. 2A)), resulting in the received and image formation in FIGS. 2B, 2C. The focus is on the specimen 1 so as to produce the final spots 52, 52'. In particular, FIG. 2B shows the resulting 4x5 (simplified) grid for the resulting confocal illumination area to guide the fluorescent spots 52 shown to be imaged by the charge binding detector 18. Therefore, one column is represented).

More specifically, each sublens 4'provided by the microlens array 4 has a focal plane designed to focus the light of its collected amount of light source 2 (shown within the dotted ellipse). In line with 55 (shown within the dotted ellipse), the focal plane 55 is configured with a lens diameter suitable for collecting all light directed by the microlens array 4'in this embodiment embodiment. Corresponds to the focal plane for the relay lens 12 made. However, while the relay lens 12 is useful, embodiments herein can also include any compound optical arrangement known in the art that results in all desired effects. In conclusion, the wavefront by the beam light source 2 is changed into an array (lattice pattern) of spots (for example, 52) by each sublens 4'(lens let) configured in the microlens array 4. It serves as a separate channel oriented along the initial beam path 21.

For illustration purposes of embodiments herein, FIG. 1B shows some final fluorescent images (spots 52) received, which are illuminated by a 4x5 grid of subbeams 50. (See FIG. 2A). However, the number of sub-beams 50 provided by a particular Microlens Array 4 is not limited to only 20 grids (ie, frameworks), but at least 2 to 20, and more than 20 if desired. It can include several grid frameworks.

For an even better understanding of the microlens array 4, each configured sublens 4'forms a separate channel to act as a new light source. The individual separate channels are spatially separated to avoid interference effects (crosstalk) due to the same array configuration. The result is, in the end, separate and unique illuminated areas, resulting in separate imaging channels, acting like multiple confocal microscopes located adjacent to each other. Thus, after passing through the microlens array 4, adjacent separate channels oriented along the beam path 21 will eventually illuminate the specimen 1 at a unique site by the objective lens 14. Received and redirected to the dichroic component 8 (often a long-pass dichroic mirror for fluorescent applications) by a steering mirror 6 (eg, a piezoscanning mirror) so that it can be received and oriented. ..

In the embodiment of the fluorescence example, the test body 1 used to demonstrate the practical embodiment was labeled with a red dye that fluoresces in the range of 600 to 750 nm. Fluorescence will be described for this illustration, but it should also be understood that reflections from the target of the specimen 1 (ie, the region illuminated by the light source 2) can be utilized if desired. Thus, with respect to Specimen 1 irradiated with light at a plurality of unique targets provided by a grid of subbeams 50 at an excitation wavelength (eg, 532 nm), the emitted light substantially from this particular dye is, for example, FIG. As shown by the image of 1B, the result was induced fluorescence between 600 nm and 750 nm. The induced emission light is then recollected by the cofocal geometric structure of the microscope objective lens 14, and then the emitted light beam further attenuates a particular emission band in order to mitigate unwanted signals with the dichroic mirror 8 and It was reoriented by the relay lens 12 to pass through the filter 17 (eg, edge filter, notch filter, etc.). An acceptable emission band (eg, 600 nm-750 nm) that passes through the filter 17 is then preferably imaged on the CCD (shown as a grid spot in FIG. 1B) and is often usually usually. Located in a plane accommodating a hard pinhole opening in a standard confocal microscope type geometry.

With respect to the dichroic mirror 8, the optical components are in a fixed mode of operation (ie, designed for specific reflection and transmission optical frequency bands), while single-parameter fluorescence and sophisticated multi-channel image formation. In order to provide better convenience without changing the dichroic mirror 8 when the mode of operation is more preferred by any of the embodiments herein, the dichroic mirror 8 and further the filter 17 are operator / consumer. If desired, it can be replaced with an acoustic-optical beam splitter (AOBS) and associated drive electronics (not shown). In such an exemplary configuration / operation, the user uses the controller / processor 102 to select a set of colors for excitation from a particular light source 2. The controller / processor 102 is then programmed to automatically direct the line onto the specimen 1 and, in the case of this non-limiting embodiment, to deliver the desired fluorescence emission. , AOBS can be operated. In addition, such AOBS components can also be utilized for reflection arrangements. Thus, in this embodiment, for example, using different illumination wavelengths (eg, using a tunable laser or a high intensity white light source), the various luminescent fluorophores are rapidly image-forming without harmful delays. Can be done.

As another exemplary arrangement, such an array of image-formed spots 52 can then be accurately approximated to a Gaussian function. Another option to emulate the behavior of traditional CLSM is to define a virtual pinhole at each array point. For example, a retractable hard pinhole arrangement (not shown) that is designed to match the grid pattern of light irradiation is available, but the virtual pinhole at each array point is a collection of virtually all photons. It is especially useful in fluorescent arrangements where all photons are required.

In particular, the CCD can be configured to operate as multiple virtual synthetic pinholes, thereby improving the signal-to-noise ratio (SNR) in high-throughput schemes by using a rapid CCD time acquisition architecture. In addition, the maximum number of emitted photons can be collected. If the signal-to-noise ratio is still inadequate, the illumination power of light source 2 can be increased and / or better light sensitive cameras such as EMCCD and sCMOS technology can be used. For example, sCMOS technology often has 5.5 megapixels with even higher resolution, not only allowing a (larger) larger field of view, without compromising read noise, dynamic range, or frame rate. Includes sensor. Moreover, such sensors can reduce read noise to 1.3 electron rms to achieve 100 full frames per second, all with any embodiment disclosed herein. It is used beneficially.

With reference to FIG. 3A again, the embodiments shown show further details of the components of a modular scanning confocal optical microscope (COP) with digital image processing capability, as generally referred to by number 300. It is the composition of another example that accompanies. The description of FIG. 3A will outline the components in the configuration, and further details, in the embodiments described below.

Therefore, as shown in FIG. 3A, the digital confocal light microscope 300 often (because it includes a photodetector configured as a CCD 312) has an illumination source 302 and one or more photodetectors 306. And 308. Other components include an objective lens 314 and a dichroic beam splitter 318 (to include AOBS as described above, if desired). In certain embodiments, the digital cofocal optical profile microscope 300, as needed, at least of the x-axis, y-axis, or z-axis, as described above for FIG. 1A. A translational moving stage configured to carry and move the test piece 1 with respect to the objective lens 314 can be included along the 1.

The digital confocal light microscope 300 includes all controllable components such as illumination sources 302, light detectors 306 and 308, and / or configured movable stage components (eg, PZT stage 311 and parallel movement). It can also include a controller / processor 102 as described above, which is operatively coupled to, for example, stage 316). In another embodiment, the digital confocal optical profile microscope 300 is further configured to focus the scanning mirror and / or the light irradiation on the specimen 1 at a particular position (x, y). Other suitable optical components can also be included. In a further embodiment, the digital confocal optical profile microscope 300 further comprises a frame, eyepieces, diaphragm, and / or other suitable mechanical / optical as described in some detail in the following examples. Components can also be included.

The illumination source 302 (as also described above for embodiments of FIG. 1) is an electromagnetic source, eg, but not limited to, a laser, a light emitting diode, a laser diode, a halogen lamp. , Mercury lamps, and / or other suitable types of light sources configured to generate illumination beams (not shown) with the desired optical properties, and the like. Illumination beams, as will be appreciated by those skilled in the art, are often spherical, but can be manipulated to have beam shape parameters that are flat and / or can also include other suitable profiles. ..

The beam splitter 318 (for example, to include the AOBS) is positioned to receive a beam for illumination from the illumination source 302. As is the preferred mode of operation, the beam splitter 318 uses the objective lens 318 with the illumination beam (not shown) provided by the illumination source 302, as also described above for FIG. 1A. Is configured to be directed towards the test piece 1. The beam splitter 318, as described above for FIG. 1A, includes an objective lens 318 and / or any other intermediary optical component such as a tube (relay) lens (not shown). Also positioned to receive the reflected beam from test piece 1 and / or the fluorescing beam (not shown) as directed by. The beam splitter 54 then splits the received beam (reflected beam and / or fluorescing beam) into a photodetector CCD array 312, and / or a beam that emits fluorescence, as described in detail in the embodiments below. / Or configured to orient towards any other optical detector element (eg, photodetectors 306, 308).

The beam splitter 318 is an uncoated optical component and a coated splitter (eg, an electron beam-deposited layered coating splitter to provide multi-wavelength selectivity (dycroic), or any layered coating optic). It can include cube splitters, half mirrors, prisms, and / or other suitable components. As described above, if desired, the beam splitter 318 can be a configured acoustic and optical beam splitter (AOBS).

The digital confocal optical profile microscope 100 is a standard confocal arrangement that directly detects the optical information of the signal beam because of the presence of the CCD in a novel mode where pinholes would typically be formed. It should be understood that it does not necessarily include physical pinholes. Because the CCD ₁ 312 array includes, for example, complementary metal oxide semiconductor (“CMOS”) detectors, EMCCD and sCMOS technologies, as described above, often as described above. Includes any desired two-dimensional charge-shifting array.

The charge-coupling detector (CCD) 312 in FIG. 3A is a detection designed to be equal to or greater than the cross-section of the received beam signal, as similarly described above for the embodiment of FIG. 1A. It can be configured by area. In many cases, the area itself for the detector is at least 5 times larger than the area of the received beam signal. The charge-shift detector (CCD) 312 is configured with a motorized control (eg, stepper control (not shown)) to move along at least one of x-axis, y-axis, or z-axis. The charge-shift detector (CCD) 312 may be able to maximize the image formation of the test piece 1 in the confocal method. However, it can also be placed away from the focal imaging plane designed by System 300 for other desired techniques. As a result, the percentage of detection areas of the photodetectors 90 and 70 filled with the signal beam is set to target values (eg, about 0.01, about 0.02, about 0.03, about 0.04, about 0. It may be increased to 05, about 0.06, about 0.07, about 0.08, about 0.09, or about 1.0).

In a further embodiment, the digital confocal optical profile microscope 300 may be configured to operate under confocal mode and / or confocal profile mode. In such a embodiment, the two modes can be operated independently or simultaneously (eg, in parallel). For example, the digital confocal optical profile microscope 300 may include retractable pinholes (not shown). Under confocal mode, retractable pinholes can be positioned between charge-coupled detectors (CCDs) 312 for filtering or at least reducing out-of-focus signals. Alternatively, under confocal profile mode, the retractable pinhole may be removed from the optical path between the charge-coupling detector (CCD) 312 to detect the profile of the signal beam.

Another very useful arrangement that can be incorporated into any modular confocal microscope disclosed herein is shown in FIGS. 3B and 3C. In general, without being bound by theory, and by using the confocal microscope 300 for exemplary purposes of the art, the input light source (eg, laser light source 302) used for light irradiation / image formation is Any using an objective lens 314 and, if appropriate, for example manual steering 326, 327, to initiate image formation of the specimen / sample that is desired to be optically investigated / adjusted. It can be oriented slightly on a non-identical line (eg, up to about 1 degree) with respect to the optical axis of the other collected / focused optics (represented as a broken line).

Due to such a configuration in relation to FIG. 3B, the sample was originally imaged, but then translated at distance Z (see plane shown as T) and / or tilted. Plane (I) will result in an image showing intensity loss (eg, due to defocus), as well as the number of shift pixels in the configured CCD 312 array (see FIG. 3C) (FIG. 3C. Also shown in (see)) for translation at (x, y). Such novel techniques are beneficially used by the microscopy configurations herein to maintain focus and mitigate data acquisition imperfections. Particularly beneficially, if for some reason the first image-formed site in the sample is translated (eg, in a plane that moves in the Z direction with respect to the confocal microscope focusing object), or during scanning. If the surface of the area to be scanned is not smooth, such events may be automatically corrected to maintain proper focus. Therefore, such events can often be imaged in real time by monitoring pixel offsets and signal intensity degradation due to defocus while the microscope is operating.

The present disclosure has been provided in general, but the same is presented for illustration purposes unless otherwise stated and is not intended to limit the disclosure, and is more easily understood by reference to the following examples. Will be.

● Example The light source module was a collimated laser diode 302 of 4.5 mW at a wavelength of 532 nm. A beam attenuation unit made of Gran-Taylor modulator P1 323 and half-wave plate 324 is used to regulate the incident laser output. The Kepler-type beam magnifier 325 magnifies the laser beam so that it fills slightly above the back aperture of the microscope objective lens 314 (Zeiss LD Plan-Neo Fluor 20 × / 0.4 Corr). A confocal pinhole (not detailed) with a diameter of 50 μm is inserted at the internal focal point that acts as a spatial filter. The magnified beam is then passed by a first beam steering mirror 326 and a second beam steering mirror 327, often through a first density filter 329 and a beam splitter 318 cube (or dichroic mirror, eg, AOBS). It is guided to the main microscopic column. The reflected (or emitted) light is then directed to the camera detection modules 400, 500 and fiber optic detection modules 600 by various beam splitter cubes 334, 335.

The camera module 400 includes an aligned detector CCD ₁ 312 (The Imaging Source, DMK 23U618 black and white camera) in a vertical arm to collect reflected and / or emitted light (eg, fluorescence) patterns. , The pattern is often stored as a 640 × 480 resolution bitmap for later image processing. The 532 nm line filter 337 (or notch filter) is positioned in front to allow only the reflected laser light to pass through. The camera module 500 includes an arrayed detector CCD ₂ 312'(The Imaging Source, DFK 23U274 color camera) in a horizontal arm used for wide field inspection and initial positioning, as well as laser light entering the camera. A 532 nm notch filter 339 (or line filter) is used to prevent this. A 200 mm focal length tube lens 342, 343 is used on both arms.

The fiber detection module 600 uses a 4x Olympus microscope objective lens 348 to connect the light to the multimode fiber 350 with a diameter of 25 μm, which is a confocal pin for the transmitted light collected by the photodetector 351. It functions as a hall. The motion module uses a piezoelectric objective scanner (Physik Instrument, PIFOC1 P725.4CD) for vertical scanning and a piezoelectric nanopositioning stage (Physik Instrument, P-611.2S) for horizontal scanning (Physik Instrument, P-611.2S). Both are shown as 352). A powered scan table (Physik Instrument, KT-120) and a 3-axis manual stage (generally both represented by 316) are used for translation of large areas. In addition, shearing interferometer 356 is used for beam collimation and intensity monitoring by photodetector 360. If appropriate, additional optical dimming filters (eg, 365, 366) are positioned.

In many cases, but not always, sample scanning is used instead of laser scanning because the design is optically simple and susceptible to optical aberrations. In addition, the field of view (FOV) of the microscope that scans the sample can be independently selected without changing the optical design of the microscope. Virtual pinholes (using CCD ₁ 312 and / or CCD ₂ 312') can be generated by selecting a region of interest (ROI) on the recorded image. The ROI contains only the pixels corresponding to the diffraction-limited spots on the sample surface according to the confocal principle. In many cases, as described below, N × N pixel square pinholes are used as virtual pinholes (synthetic openings) with N value selection, also described below. The image is analyzed to bring out additional contrast beyond the virtual pinhole. After the scan is complete, the recorded images are saved in the same order as their spatial coordinates. A computer program is then used to deconvolution the information from the recorded images.

● Two-dimensional scan A 3 mm × 3 mm chip with a total scan point of 200 × 200 at a process size of 0.5 μm along both X and Y to illustrate working modes using embodiments as described above. A two-dimensional scan of a 100 μm × 100 μm area on the carrier test piece 1 was started. _A final image covering the ROI of N × N pixels on the image recorded by the CCD 1212 was obtained. The Nyquist-Shannon sampling theorem requires at least two pixels per resolvable element. First, a 2x2 pixel square pinhole was selected. Additional cropping sizes (N = 6, 11, 21, 31, 41, 51, 101) were used to identify the optimal pinhole size for image formation. For comparison, a fiber optic module was used to represent standard confocal signal detection. A multimode fiber 350 with a diameter of 25 μm was used to collect reflected light from the same field of view.

As a method of the embodiment utilizing the microscope 300, a calibration process is often performed before the specimen 1 is scanned. This step is performed to ensure that the fiber facet and CCD are on the conjugate plane corresponding to the focal plane of the objective lens.

FIG. 4 shows a measurement axis response curve of an embodiment of an optical fiber response curve (represented as a fiber), as well as a CCD-based detection pixel cropping size (actual pixel composite aperture size N = (6). , 11, 21, 31, 41, 51, 101)) are also shown. These relatively symmetric plots share a common axis zero / focus (at Z-0) to allow the user confidence that the axis zero / focus is optically aligned with respect to the same conjugated plane. do. It should be noted that the fiber optic response curve (fiber) is relatively wider than the response curve in CCD plots up to 31x31. In the CCD plot, the width of the fiber optic response curve increased with crop size. The full width at half maximum (FWHM) is 7 μm when N = 2 and 25.72 μm when N = 21. Above N = 31 × 31, no clear peak is found.

An intensity map revealing the geometric profile of Specimen 1 from a fiber optic confocal scan is shown below for the polarity marker (O), width marker AB with respect to the top electrode (TE), And as a width CD with respect to the upper electrode (BE), it is shown in FIG. In particular, the embodiment of FIG. 1 can similarly image such a test piece 1 with the same result, but FIG. 5 shows an image of a chip carrier test piece 1 using the embodiment of FIG. ..

The CCD-based detection results are shown in FIGS. 6A-6I. In particular, FIG. 6A shows the image formation of the fiber, FIG. 6B shows the crop size of 2 × 2N pixels (synthetic aperture), and FIG. 6C shows the crop size of N = 6 × 6 pixels (synthetic aperture). 6D shows a crop size of N = 11 × 11 pixels (composite opening), FIG. 6E shows a crop size of 21 × 21N pixels (composite opening), and FIG. 6F shows a crop size of 31 × 31N pixels. (Synthetic opening) is shown, FIG. 6G shows a crop size of 41 × 41N pixels (composite opening), FIG. 6H shows a crop size of 51 × 51N pixels (composite opening), and FIG. 6I shows 101 ×. The crop size (composite opening) of 11N pixels is shown. The image has the best or acceptable resolution clarity from a 2x2 pixel array size to a 21x21 pixel array synthetic aperture, which is also consistent with the results of the response curve in FIG. It is important to keep in mind. However, while the 2x2 pixel array size to the 21x21 pixel size array (ie, synthetic aperture) is useful, the 6x6 case (see, eg, FIG. 6C) is the 2x2 case. It is important to note that it gives the closest match (see, eg, FIG. 6B) and still maintains a significant amount of pixels.

Therefore, as repeated below, a 6x6 array of pixels (see, eg, FIG. 6C) has the optimum pinhole size preferred for 3D scans that require more complex analysis and best performance. However, 21x21 (see, eg, FIG. 6E) is a pinhole array size for 2D scanning that requires meaningful comparisons between fiber and CCD experiments and low computational load. Is. Also noteworthy is the manual control of PI-611. When the laser spot disappears at the side edge of the inspection camera CCD ₂ , the electrode width AB is shown in FIG. 5 in a 2S piezoelectric stage for translating over the width of the upper right (TE) and lower left (BE) electrodes. And got a visual evaluation of the CD. That is, AB is 28-29 μm and CD is 27-30 μm.

As an objective approach, which is an alternative embodiment, a computer program to calculate the mean width of the upper right and lower left electrodes, AB (TE) and CD (BE), instead of the decoupling and dispersion of the selected line pairs. It was used. Such a program first calculates the thresholds for completely separating the gold-plated electrodes and markers from the substrate, and then calculates the width of the electrodes above these thresholds at each X or Y position.

With reference to FIGS. 7A, 7B, and 7C again, FIG. 7A includes a variety of polarity markers to indicate the horizontal width of the polarity marker O (see FIG. 5) at a particular Y position. Showing a strength profile of a horizontal line penetrating a pinhole-sized electrode (AB), FIG. 7B is an equipolar marker to indicate the vertical width of the polar marker O (see FIG. 5) at a particular X position. And the intensity profile of the vertical line through the electrodes (AB) of various pinhole sizes, while FIG. 7C shows the case of an optical fiber and the case of a CCD with a pinhole size of 21 × 21 pixels. The full width profile of the electrodes over the measured X and Y ranges is shown. As a result, a series of width values is obtained.

The upper half of FIG. 7C represents the maximum horizontal width of the electrodes at each Y position. The lower half of FIG. 7C represents the maximum vertical width of the electrodes at each X position. The intensity profiles of the horizontal and vertical lines penetrating the center of the polarity marker (O) in FIGS. 7A and 7B show a decrease in contrast and an increase in noise with increasing pinhole size. The diffraction-limited spot diameter in the focal plane of the objective lens (eg, Zeiss LD Plan Neofluor 20 × / 0.4 Corr) is 1.62 μm at 532 nm. Diffraction-limited spots are projected by a digital confocal optical microscope 300 optics onto an area with a diameter of 39.56 μm on the CCD, surrounded by 7 × 7 pixels. The projected area on the fiber facet 350 in the optical fiber detection arm 600 is 8.9 μm in diameter. Therefore, a multimode fiber 350 core with a diameter of 25 μm surrounds a 2.81 × Airy unit (AU) corresponding to the equivalent surface area of 20 × 20 pixels of the CCD.

The case of N = 2 × 2, which is the limit size of point detection by the CCD (see, for example, FIG. 6B), shows the minimum width of the deviation in the width measurement. Larger pinhole sizes, 6x6 (see, eg, FIG. 6C) and 11x11 (see, eg, FIG. 6D), show slightly larger width values, but the 2x2 case. Maintain a standard deviation value comparable to. The case of N = 21 × 21 (see, eg, FIG. 6E) shows a slight increase in noise. Beyond N = 31 × 31 (see, eg, FIG. 6F), the noise level increases significantly and the resolution is even more rounded, as evidenced by the fact that the end profile of the marker in the lower right position is even more rounded. descend. The case of N = 101 × 101, which corresponds to wide-field image formation, shows the largest standard deviation and the highest noise level.

The increased noise in the case of larger pinholes is believed to be the result of inclusion of pixels that do not receive more photons, and these pixels are subject to read noise and dark current noise outside the diffraction-limited spot. It is exposed and thereby increases the overall noise level of the reconstructed image. The fiber optic case shows higher numbers due to its modest core diameter size. In theory, tiny pinholes can give the best spatial resolution, but even such tiny pinholes can actually reject photons that could be used to counter noise. Should be understood.

However, as mentioned above, the 6x6 case (see, eg, FIG. 6C) gives the closest match to the 2x2 case (see, eg, FIG. 6B), and, It still maintains a significant amount of pixels. Therefore, 6x6 (see, eg, FIG. 6C) is the preferred optimum pinhole size for 3D scans that require more complex analysis and best performance, while 21x21 (eg, FIG. 6E). Is a preferred pinhole size for 2D scanning that requires a meaningful comparison between fiber and CCD experiments and a low computational load.

● Permanent light conduction Here, the process of processing / adjusting materials exhibiting permanent light conduction is targeted. Annealed strontium titanate (SrTiO ₃ or STO) single crystals exhibit permanent photoconductivity (PPC) at room temperature. For example, irradiation with subgap light using the modular confocal embodiment shown in FIG. 3A reduces resistance by three orders of magnitude, which lasts up to one year or longer. The results of IR spectroscopy and two-point resistance measurements show that water vapor at 1200 ° C. results in a group of hydrogen and oxygen pores resulting in large PPCs. Deuterium substitution experiments demonstrated the formation of dihydrogen centers after exposure to light. Therefore, by sub / bandgap light (at least 2.9 eV (eg, 450 nm)) managed using the confocal arrangement disclosed herein, the substituted hydrogen leaves the oxygen moiety and is metastable OH. Form a bond.

In particular, certain annealing treatments induced permanent photoconductivity (PPC), in which case the sample treated with light with an energy of 2.9 eV or higher (test piece 1) transitioned from insulating to conductive. .. The change in conductivity is three orders of magnitude, occurs at room temperature, and is stable for over a year. A surprising and unexpected increase in conductivity was confirmed by electrical measurements (see Figure 8) as well as increased free carrier absorption in the infrared (IR) region of the spectrum.

As a novel and informative embodiment, for example, using the modular confocal arrangement disclosed herein, the exact conduction path to the diffraction limit spot size of the objective lens is drawn by light on the resistance sample. Can be done. Specimen 1 can be monitored (image forming) using the automatic focusing means disclosed above to ensure accuracy with respect to the desired adjusted area. Therefore, the process using the embodiments of this specification induces PPC, i.e., conductivity. When the conductivity increases, the charge on the surface of the material increases to include some other semiconductor material (eg, gallium nitride). Such a process can combine PPC technology with optical lithography applications and other semiconductor applications, such as including the deposition of cationic conductive materials (eg, Ag ⁺¹ ) at the conditioned site. Additional applications include bioelectronics applications for PPC conditioning of semiconductor substrates, where desired cells with a particular charge will adhere to the surface of the conditioned PPC material.

FIG. 8 shows a two-point resistance measurement of the STO sample, which had a resistance of about 1 MΩ before exposure. After irradiation with 405 nm light, the resistance dropped to about 1 kΩ. Resistance was measured in the dark for at least 1 year. The data were based on experiments and approximated to the sum of the two exponential functions using time constants of 17 days and 800 years. The long-term behavior, surprisingly and unexpectedly, shows that the resistance change is essentially permanent at room temperature.

To achieve a large PPC, the SrO powder is placed in a sealed ampoule with the STO sample and annealed at 1200 ° C. under vacuum. Annealing under vacuum prior to the "SrO" annealing process is often a useful step for preparing crystals. The vacuum annealing process introduces oxygen vacancies to induce PPC. The presence of hydrogen and oxygen during the annealing treatment at 1200 ° C. is beneficial for PPC.
Again, the following examples are provided for illustration purposes only and are not intended to be the limitations of this disclosure unless otherwise stated.

● Example ● Experimental method The annealing method for inducing PPC in STO involves encapsulating a bulk single crystal of STO in a molten silica ampoule together with 0.5 g of strontium oxide (SrO) powder under low vacuum. The sample space in the ampoule has a length of about 7.6 cm and a diameter of 1.6 cm. Ampoules were annealed in a horizontal tube furnace at 1200 ° C. for 1 hour. The sample was taken out immediately and cooled in the dark under ambient air for about 10 minutes. The IR spectrum was obtained using a Bomem DA8 vacuum Fourier transform infrared (FTIR) spectrometer. A mercury cadmium tellurium (MCT) detector was used to obtain powder spectra at room temperature. Cold spectra were obtained using an antimonide indium (InSb) detector and a Janice closed cycle helium cryostat at a resolution of 1 cm ^-1 .

A 405 nm light emitting diode (LED) was placed in the cryostat to allow exposure without moving the sample or breaking the vacuum. Anhydrous Sr (OH) ₂ and SrO powders were purchased from Sigma-Aldrich. Deuterated strontium hydroxide Sr (OD) ₂ was prepared by placing SrO powder and heavy water (D2O) in a sealed container (humidity chamber) for ₂ days. D ₂ O formed heavy water vapor in the same humidity chamber, which was easily absorbed by strontium oxide to form Sr (OD) ₂ . The mass of the powder before and after putting it in the humidity chamber (1.0 g) and after (1.8 g) was measured. The corresponding increase in mass indicated that the powder absorbed about ₄ D2O molecules per SrO.

● Results
● SrO powder
The presence of SrO powder during the annealing treatment at 1200 ° C. was found to be important for large PPCs. Other ambient conditions, such as annealing in Ar without any powder, resulted in a highly n-type material. The annealing treatment at 1200 ° C. was carried out using the newly obtained SrO powder. The sample was not in a high resistance state after annealing and before exposure, but rather exhibited conductivity (about 300 times due to the two-point pressurized indium contact). This indicates that the SrO powder must be aged in the surrounding atmosphere to obtain the optimum PPC.

For sufficient transparency, SrO powder was mixed with KBr and pressed to pellet. Materials from freshly opened bottles were compared to materials exposed to the atmosphere for 6 days. Several additional IR peaks appear in air-exposed samples (see Figure 9), where the IR peaks are strontium hydroxide Sr (OH) ₂ or Sr (OH) ₂ nH ₂ O, In addition _, it belongs to strontium carbonate SrCO3. The CO ₃ ^2- anion has a wide absorption band centered around about 1445 cm ^-1 , which is believed to be the result of asymmetric stretch vibration, while the 866 cm ^-1 and 599 cm ^-1 lines bend vibration. Is. The peak of 3590 cm ^-1 belongs to the expansion and contraction mode of OH ^-1 in Sr (OH) ₂ , while the broad absorption centered around about 2835 cm ^-1 is H ₂ in Sr (OH) ₂ nH ₂ O. It is considered to belong to the expansion / contraction mode of O. This indicates that SrO absorbs water and carbon dioxide, which can be released during the hot annealing process.

● Strontium hydroxide
● Evidence for PPC
Strontium hydroxide is a pollutant species in SrO powder. To test the effect of strontium hydroxide on PPC, a small amount (0.1 g) of anhydrous Sr (OH) ₂ was placed in an ampoule without any SrO. The powder was located at the heatsinked end of the ampoule to prevent premature decomposition of the strontium hydroxide powder during the hydrogen-oxygen torch sealing process. Then, the sample was annealed. At 1200 ° C., Sr (OH) ₂ decomposes into SrO and _H2O .

This sample shows PPC when measured by two methods. First, exposure causes a dramatic decrease in the intensity of transmitted light passing through the sample, which corresponds to a large increase in free carrier absorption (FIG. 10). Second, the two-point resistance of the sample at room temperature using pressurized indium contacts was reduced 400-fold, as shown in Table I.

This result demonstrates that hot steam can cause PPC. To further investigate the role of water and its components, the evacuated ampoule was refilled with 0.1 g of Sr (OH) ₂ powder with either hydrogen or oxygen at about 0.5 atmospheres. Samples annealed in a hydrogen-rich atmosphere showed PPC (see Figure 10). The PPC was less dramatic than the annealing process on the exhausted ampoules and was a double digit resistance change (Table I). Hydrogen is a reducing atmosphere and can introduce more oxygen vacancies, thus reducing the resistance of the pre-illumination state by introducing more free carriers. In contrast, the oxygen-rich atmosphere did not show PPC (Fig. 10). This is consistent with the result that oxygen vacancies are important for PPC. The additional oxygen during the annealing process suppresses the formation of oxygen vacancies, resulting in a non-sensitive resistant material.

● The results of the water SR (OH) ₂ annealing treatment suggest that water vapor is also important for PPC. To further test this, STO was annealed under steam without the use of any powder. To minimize evaporation of water under vacuum, 0.04 g of water was frozen and ice was placed in a heatsink ampoule with the sample. About half of the water remains after sealing, which corresponds to a pressure of about 10-15 atmospheres of water vapor during the annealing process. This ampoule was annealed under standard conditions. The sample shows PPC, which indicates that only water vapor is the cause of PPC (FIG. 11). Hydrogen lines are similar to those observed in the Sr (OH) ₂ annealing process, except that no weak lines of 3531 cm ^-1 are observed and additional 3542 cm ^-1 accompanying lines are observed.

Table I shown above lists the annealing conditions and two-point resistance measurements before and after exposure. Deuterium-substituted samples begin in a higher resistance state prior to exposure to light. The water vapor pressure during the annealing process correlates with the resistance value observed before exposure. This is because water has a very slight oxidative effect, which will reduce the number of oxygen vacancies present. Based on these measurements, the model for PPC is: For a "hydrogen-rich" annealing treatment at 1200 ° C., the sample contains (VSr-H) and HO impurities.

Upon exposure to light, hydrogen migrates from the oxygen site, forming (VSr-2H) and emitting two electrons as follows:

This results in a decrease in (VSr- _H ) ^-IR absorption peak (HI) and an increase in sideband (VSr-2H) ⁰ . In the case of hydrogen deficiency CO ₂ annealing treatment, most Sr pores are passivated. The PPC reaction is given by:

This results in an increase in (VSr ^- H) -peaks and disappearance of the (VSr-2H) ⁰ sideband. The proposed model is one non-limiting explanation for experimental observations. Some of the OH bonds observed in IR are not due to the vacancy-hydrogen complex. They can be, for example, acceptor-hydrogen pairs. In that case, electrons will also be emitted. The point is that if H leaves its replacement site, the oxygen vacancies can function as a shallow double donor. The formed OH bond is strong enough to prevent the hydrogen atom from returning to its substitution site at room temperature.

Claims (22)

It is a modular confocal microscope,
With an electromagnetic radiation source,
A beam steering means arranged so as to orient the electromagnetic radiation source on a non-identical line with respect to the optical axis of the focusing lens means configured in the modular confocal microscope.
With at least one aligned detector configured on a composite confocal plane along the beam path to one or more target sites in the specimen.
With a processor connected to at least one of the aligned detectors configured to identify the two-dimensional and / or three-dimensional properties of one or more of the target sites of the test piece.
Equipped with
The electromagnetic radiation source focused and oriented on a non-identical line is used for the imaging basis of one or more of the target sites in the specimen.
The aligned detector is configured to provide automatic correction information in order to maintain the focus and image quality of one or more of the target sites using the image forming basis.
The aligned detectors provide high throughput constituting a synthetic aperture at least in the pixel range of an array of N = 2 × 2.
A modular confocal microscope characterized by this. The configured synthetic aperture is in the pixel range from an array of N = 2 × 2 to an array of N = 101 × 101.
The modular confocal microscope according to claim 1. The configured synthetic aperture is in the pixel range from an array of N = 2 × 2 to an array of N = 21 × 21.
The modular confocal microscope according to claim 1. A steering mirror or xyz stage connected to the processor is oriented to scan the specimen.
The modular confocal microscope according to claim 1. At least one of the aligned detectors is a two-dimensional aligned charge-shift detector (CCD).
The modular confocal microscope according to claim 1. The two-dimensionally aligned charge-bond detector (CCD) is selected from complementary metal oxide semiconductors (CMOS), EMCCD, or sCMOS.
The modular confocal microscope according to claim 1. A beam splitter selected from a long-range dichroic mirror beam splitter, a cube splitter, a half mirror, a prism, and an acoustic optical beam splitter (AOBS).
The modular confocal microscope according to claim 1. With an electromagnetic lighting source,
A microlens array arranged to receive the electromagnetic illumination source, and
Optical lens means configured to collect and focus multiple subbeams on multiple target sites,
Image formation of the resulting multiple sites by the reflected reflected light configured along the second beam path and / or the emitted light provided by the signals from the plurality of focused subbeams. With at least one aligned detector configured to
With a processor connected to at least one of the aligned detectors configured to identify the 2D and / or 3D characteristics of the target site.
Equipped with
The received electromagnetic illumination source is reconstructed by the microlens array into a plurality of the sub-beams running adjacently along the first beam path.
The optical lens means collects and focuses the plurality of the sub-beams on the plurality of target sites, and then collects the induced reflected light and / or the emitted light from each of the plurality of target sites. Then, orient along the second beam path,
The aligned detectors provide high throughput constituting a synthetic aperture at least in the pixel range of an array of N = 2 × 2.
A modular microlens array confocal microscope characterized by this. The configured synthetic aperture is in the pixel range from an array of N = 2 × 2 to an array of N = 101 × 101.
The modular microlens array confocal microscope according to claim 8. The configured synthetic aperture is in the pixel range from an array of N = 2 × 2 to an array of N = 21 × 21.
The modular microlens array confocal microscope according to claim 8. A beam steering means arranged so as to direct an electromagnetic radiation source on a non-identical line with respect to the optical axis of the optical lens means, and a beam steering means.
With at least one of the aligned detectors configured on a composite confocal plane along the beam path to one or more of the target sites in the specimen.
Equipped with
The electromagnetic radiation source focused and oriented on a non-identical line is used for the imaging basis of one or more of the target sites in the specimen.
The aligned detector is configured to provide automatic correction information in order to maintain the focus and image quality of one or more of the target sites using the image forming basis.
The modular microlens array confocal microscope according to claim 8. A beam splitter selected from a long-range dichroic mirror beam splitter, a cube splitter, a half mirror, a prism, and an acoustic optical beam splitter (AOBS).
The modular microlens array confocal microscope according to claim 8. A method of adjusting a material using a modular confocal microscope to induce permanent photoconduction (PPC).
The process of incorporating a modular confocal microscope to simultaneously image and adjust one or more target sites in a specimen exhibiting permanent light conduction, and
The process of selecting a predetermined emission spectrum provided by an electromagnetic radiation source, and
The step of irradiating the initial contact point through the confocal geometry of the modular confocal microscope,
A step of translating the test piece so as to draw a route of exposure of the test piece to the irradiation while maintaining the irradiation.
Including
The modular confocal microscope
With the electromagnetic radiation source
A beam steering means arranged so as to orient the electromagnetic radiation source on a non-identical line with respect to the optical axis of the focusing lens means configured in the modular confocal microscope.
With at least one aligned detector configured on a composite confocal plane along the beam path to one or more of the target sites in the specimen.
With a processor connected to at least one of the aligned detectors configured to identify the two-dimensional and / or three-dimensional properties of one or more of the target sites of the test piece.
Equipped with
The electromagnetic radiation source focused and oriented on a non-identical line is used for the imaging basis of one or more of the target sites in the specimen.
The aligned detector is configured to provide automatic correction information in order to maintain the focus and image quality of one or more of the target sites using the image forming basis.
The aligned detectors provide high throughput constituting a synthetic aperture at least in the pixel range of an array of N = 2 × 2.
A method of adjusting a material using a modular confocal microscope. The configured synthetic aperture is in the pixel range from an array of N = 2 × 2 to an array of N = 101 × 101.
A method of preparing a material using the modular confocal microscope according to claim 13. The configured synthetic aperture is in the pixel range from an array of N = 2 × 2 to an array of N = 21 × 21.
A method of preparing a material using the modular confocal microscope according to claim 13. The test piece showing permanent light conduction is an oxide,
A method of preparing a material using the modular confocal microscope according to claim 13. The test piece is at least one material selected from strontium titanate (SrTiO ₃ ) and zinc oxide (ZnO).
The method of preparing a material using the modular confocal microscope according to claim 16. The test piece exhibiting permanent photoconductivity is a group III-V semiconductor material selected from gallium nitride (GaN), gallium nitride indium arsenide (GaInNAs), and gallium arsenide (GaAs).
A method of preparing a material using the modular confocal microscope according to claim 13. The material exhibits permanent photoconductivity (PPC) for at least one year.
A method of preparing a material using the modular confocal microscope according to claim 13. The material is annealed up to 1200 ° C.
A method of preparing a material using the modular confocal microscope according to claim 13. The material is annealed under steam.
A method of preparing a material using the modular confocal microscope according to claim 13. The step of attaching a desired biological cell having a specific charge onto the surface of the prepared PPC material comprises the step of attaching.
A method of preparing a material using the modular confocal microscope according to claim 13.

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